The contamination of water with human enteric viruses continues to present a serious threat to the public health in many countries, including the United States. Enteric viruses can cause debilitating diseases, and many outbreaks result from contaminated drinking water. For example, from 1971 to 1999, 9% of reported outbreaks of infectious diseases associated with ground water contamination were attributed to viral agents. In 1982, 73 cases of hepatitis A virus (HAV) were documented to be the result of consuming contaminated water from a single spring in Meade County, Ky. (See Bergeisen, G. H.; Hinds, M. W.; Skaggs, J. W. Am. J. Public Health 1985, 75, 161-164). Norwalk virus and Norwalk-like viruses (noroviruses) are regarded as major causes of foodborne and waterborne viral gastroenteritis. Outbreaks of viral gastroenteritis have been associated with contamination of water supplies, raw foods, and food products prepared by ill food handlers. In some instances, outbreaks of norovirus gastroenteritis have been linked to the failure of a septic tank system (See, Bellar M.; Ellis, A.; Lee, S H.; Drebot, M. A. et al. JAMA 1997, 278, 563-568).
Conventionally treated drinking water may still contain human enteric viruses and cause outbreaks even when those waters have met water quality criteria based upon coliform bacteria densities and turbidity. Furthermore, because enteric viruses are generally infectious at relatively low concentrations, the detection of virus samples in environmental waters typically requires the collection of large sample volumes such as, for example, more than 1,000 liters.
Adsorption-elution is one known method used to concentrate virus samples using large volumes of water. Adsorption-elution methods rely upon electrostatically (negatively or positively) charged microporous filters to capture virus particles. In particular, virus particles are negatively charged at pH 7.0 and adsorb to the filter as the water sample passes through the electrostatic filter. Although inexpensive, microporous filters having negatively charged materials such as nitrocellulose (Millipore HA), fiberglas (Filterite), and cellulose (Whatman) typically require extensive pre-conditioning of the water to facilitate binding interactions between the negatively charged surface material and negatively charged virus particles (See, Lukasik J.; Scott, T. M.; Andryshak, D.; Farrah, S. R. Appl Environ Microbiol. 2000, 66, 2914-20).
Pre-conditioning treatments of the water may include acidification of the water sample or the addition of multivalent cationic salts such as, for example, magnesium chloride or aluminum chloride to serve as bridging molecules between the negatively charged microporous filters and the negative charged viral particles to facilitate virus capture. In such pre-conditioning treatments, the addition of multi-valent cations effects a charge reversal on the negatively charged viral particles, providing for electrostatic binding interactions on the negatively charged microporous filter surface.
Alternatively, the addition of multi-valent cations with the negatively charged microporous filter provides counter-ion flux water on an ion-selective membrane (e.g., a filter), which produces a thick positively charged double layer near the membrane surface. (See, Ben, Y.; Chang, H.-C., J. Fluid Mech. 2002, 461, 229-238). This external double layer has a net charge higher than that of the membrane, thus effectively reversing the charge of the membrane to create a virus binding surface. However, this alternative positively charged double layer mechanism can only occur if the pores of the membrane filter are sufficiently small such that electro-osmotic flow and neutralizing convection are minimized, and only ion-selective electro-migration drives a cation flux into the membrane. As a result, negatively charged membrane filters having small pore sizes (e.g., 0.2 to 0.45 μm) often times minimize internal flow, which may severely restrict the volume of sample that can be filtered.
Electropositive microporous filters, on the other hand, have a positively charged surface that facilitate the deposition and retention of the particles on the surface of the filter media. Electropositive filters typically do not require pre-conditioning treatments of the water sample and may accommodate larger sample volumes due to their large porosity (10 μm) and extensive surface area (See Sobsey, M. D.; Jones, B. L.; Appl. Environ. Microbiol. 1979, 37, 588-595 and Sobsey M. D.; Glass, J. S. Appl Environ Microbiol 1980, 40, 201-210). However, these filters are usually unsuitable for concentration of particular viruses and are relatively more expensive. The appreciable costs of these filters, coupled with erratic recoveries for some important viral agents often times preclude their routine use.
Both the negatively and positively charged microporous filters require post-filtration process to concentrate the viral particles. Post-filtration processing rely upon the use of relatively large volumes (e.g., ˜1,000 mL) of highly alkaline, protein-rich eluent, for example beef extract/glycine, that may interfere with downstream enzyme-based assay procedures such as the polymerase chain reaction (PCR) due to the high concentration of mammalian DNA in the eluents. Polymerase chain reaction (PCR) amplifies (i.e., replicates) a specific regions of a DNA strand (the DNA target) by in vitro enzymatic replication. Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. PCR detects viral DNA using primers specific to the targeted sequences in the DNA of a virus and can be used for diagnostic analyses or DNA sequencing of the viral genome.
Thus, large elution volumes also typically require secondary concentration to render the sample compatible with assay procedures that rely upon mammalian cell cultures (e.g., 20-30 mL). These secondary concentration methods include, for example, organic flocculation, adsorption-elution, etc. More recently, however, alternative elution procedures relying upon defined eluents (e.g. amino acids) have been described for the recovery of nonculturable viruses from water, with encouraging reductions in assay interferences (See Hedberg, C. W.; Osterholm, M. T. Clin. Microbiol. Rev. 1993, 6, 199-210 and Kittigul, L.; Khamoun, P.; Sujirarat, D.; Utrarachkij, F.; Chitpirom, K.; Chaichantanakit, N.; Vathanophas, K. Mem Inst Oswaldo Cruz. 2001, 96, 815-21).
Ultrafiltration is another known method used to concentrate virus samples using large volumes of water. Ultrafiltration also relies on positively charged filter media for concentration of enteric viruses (Li, J. W.; Wang X. W.; Rui Q. Y.; Song N.; Zhang F. G.; Ou Y. C.; Chao F. H. J. Virol. Methods 1998, 74, 99-108). Ultrafiltration filters have very small pore sizes that only allow small molecules to pass through the filter and retain the viruses within the circulating sample, which is recirculated until the required sample size is reached. A small pour size filter concentrates all particles, including virus microbe particles, present in the water sample, which can minimize bias in concentration of different virus types within the sample. After the required volume is reached, an elution is used to concentrate the viral particles adsorbed onto the filter membrane.
The positively charged ultrafiltration filter may be composed of, for example, NaCO3, AlCl3 and silica gel (Li et al.). Recently, hollow fiber ultrafilters have been used to detect enteric viruses in a variety of water samples and recoveries in excess of 50% have been reported (Morales-Morales H. A.; Vidal, G.; Olszewski, J.; Rock, C. M.; Dasgupta, D.; Oshima, K. H.; Smith, G. B. Appl Environ Microbiol 2003, 69, 4098-102). However, the small pores of the filter membrane typically become blocked during filtering by organic matter in environmental water samples. Additionally, these ultrafiltration filters are relatively expensive, must be pretreated to block nonspecific adsorption onto the membrane, and samples must be recirculated through filter cartridges under pressure at relatively slow flow rates (e.g., 200-300 mL/min). These requirements make it impractical to filter large volumes of water in the field.